Heat-resistant keratinase and application thereof

Abstract

The invention discloses heat-resistant keratinase, the amino acid sequence of the heat-resistant keratinase is shown as SEQ ID NO: 1, the gene is obtained from Brevibacillus gelatini LD5, heterologous expression, purification and characterization are carried out on the keratinase through a genetic engineering means, experimental data show that the keratinase has keratinase activity at 30-90 DEG C, the reaction temperature of the keratinase is as high as 90 DEG C, and the keratinase can be used for preparing the heat-resistant keratinase. High thermal stability is realized at 60 DEG C; after the keratinase BrgM4 is used for carrying out enzymolysis on chicken feathers for 48 hours at the temperature of 50 DEG C and the pH value of 6.5, the soluble keratin content reaches 8.87 mg / mL; the keratinase disclosed by the invention is beneficial to degradation of keratin waste at high temperature.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a heat-resistant keratinase and application thereof. Background Art

[0002] Keratin is a type of fibrous, stubborn, non-nutritional structural protein belonging to the superfamily of intermediate filament proteins, which constitutes most of the animal epidermal appendages. Keratin is highly insoluble due to its high sulfur content and filamentous properties. Physical and chemical treatment methods of keratin not only pollute the environment, but also seriously affect the quality of the obtained products. Keratinase (EC 3.4.-.-) is a type of enzyme that can catalyze the degradation of keratin. The use of keratinase to degrade keratin has the advantages of mild reaction, no addition of toxic chemical reagents, and no destruction of protein nutrition. Thermostable keratinase has high thermal stability. When it is used in industrial production, it can not only reduce the contamination of foreign bacteria, but also reduce the viscosity of the reactants, increase fluidity, and improve the reaction efficiency. In addition, the structure of keratin tends to be loose at high temperatures, which is more conducive to the attack of proteases. Keratinase with high thermal stability can also be used in other non-keratin degradation, such as food and animal feed production, green leather making, food processing, and laundry industries. Animal feed production involves high-temperature granulation, expansion, extrusion and steam stripping by enzymatic methods. Highly thermally stable proteases can improve the product value of animal feed. In addition, most proteases used in the washing industry are medium-temperature proteases, which are not easy to work at high temperatures, while thermostable proteases can solve this problem.

[0003] In the industrial application of enzymes, the heat resistance and thermal stability of enzymes have always been the key to restricting the industrialization of enzymes, and are also important indicators to measure the application value of an enzyme. At present, most studies have found that keratinase isolated from different microbial fermentation broths has poor heat resistance and thermal stability, and only a few studies have applied keratinase to the degradation of keratin biomass. Therefore, exploring the application of heat-resistant keratinase in high-temperature industrial environments and keratin waste degradation has become the direction of many researchers' efforts. Summary of the invention

[0004] The present invention provides a heat-resistant keratinase BrgM4, derived from Brevibacillus gelatini LD5, the amino acid sequence of which is shown in SEQ ID NO:1, the nucleotide sequence of which is shown in SEQ ID NO:2, and the molecular weight of the protein is about 34 kDa; Another object of the present invention is to apply the above-mentioned heat-resistant keratinase BrgM4 in the degradation of chicken feathers. The heat-resistant keratinase BrgM4 can degrade chicken feather waste at high temperatures.

[0005] The purpose of the present invention is achieved through the following technical solutions: 1. From Brevibacillus gelatiniThe original amino acid sequence of keratinase BrgM4 was obtained in LD5 proteomics (as shown in SEQ ID NO:1), and the nucleotide sequence corresponding to the sequence was sent to GenScript Biotech Co., Ltd. for codon optimization (the optimized nucleotide sequence is shown in SEQ ID NO:2). The sequence of BrgM4 keratinase and pET-28a plasmid (containing histidine tag) were constructed according to conventional methods to obtain a recombinant plasmid, which was transformed into Escherichia coli DH5α. After overnight culture, the clone plasmid was extracted and transformed into ArcticExpress (DE3) competent cells. Isopropylthiogalactoside (IPTG) was used to induce expression. The supernatant of keratinase expressed by IPTG was used as a crude enzyme and affinity purified using a nickel column. The purity and enzyme molecular weight of the purified BrgM4 enzyme were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified keratinase BrgM4 was obtained; 2. Enzymatic properties of keratinase BrgM4 Through temperature-pH response surface experiments, thermal stability experiments, and enzyme kinetics experiments, it was determined that the maximum reaction temperature of keratinase BrgM4 could reach 90 ℃, and it had high thermal stability at 60 ℃. m is 55.20 mg / mL, V max is 3.11 µM / min, k cat 10.37 min -1 ; 3. Experiment on the degradation of chicken feathers by keratinase BrgM4 Experiments have shown that keratinase BrgM4 can convert chicken feather keratin into soluble protein.

[0006] Advantages and technical effects of the present invention: The invention provides a heat-resistant keratinase, which is heterologously expressed, purified and characterized by genetic engineering means, and experimental data show that the keratinase has keratinase activity at 30°C-90°C, the reaction temperature of the keratinase is as high as 90°C, and it has high thermal stability at 60°C and below; after keratinase BrgM4 hydrolyzes chicken feathers for 48 h (50°C, pH 6.5), the soluble keratin content reaches 8.87 mg / mL; the keratinase prepared by the invention is helpful to provide good application prospects for high-temperature industries and provide a basis for the degradation of keratin waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The results of SDS-PAGE analysis of purified keratinase BrgM4, M: standard protein marker; 1: purified keratinase BrgM4; Figure 2 This is the time and degradation effect curve of keratinase BrgM4 hydrolyzing the standard substrate (azure keratin); Figure 3 is the response surface diagram of keratinase BrgM4 activity changing with pH and temperature; Figure 4 Fitting curve for kinetic analysis of keratinase BrgM4 (Michaelis-Menten equation); Figure 5 The results are as follows: the soluble protein concentration of chicken feathers hydrolyzed by keratinase BrgM4 (60 ℃, pH 7.5) at different time points. The data were analyzed by one-way ANOVA. The letters above the error bars represent significance. Groups with different letters have statistical differences ( p < 0.05); Figure 6 The results are as follows: the soluble protein concentration of chicken feathers hydrolyzed by keratinase BrgM4 (50 ℃, pH 6.5) at different time points. The data were analyzed by one-way ANOVA. The letters above the error bars represent significance. Groups with different letters have statistical differences ( p < 0.05). DETAILED DESCRIPTION

[0008] The present invention is further described in detail below in conjunction with the accompanying drawings, tables and examples, but the protection scope of the present invention is not limited to the contents described above. The reagents and methods used in the examples are conventional reagents and methods unless otherwise specified.

[0009] Example 1: Acquisition, heterologous expression and purification of keratinase BrgM4 1. Brevibacillus gelatini After the LD5 strain was activated, it was cultured at 50°C in a chicken feather inorganic salt medium (1% chicken feather, 0.05% NH4Cl, 0.05% NaCl, 0.03% K2HPO4, 0.04% KH2PO4, 0.01% MgCl2·6H2O, pH 7.2) for 20 h. The culture product was separated into solid and liquid to obtain the fermentation supernatant, which was sent to Beijing Novogene Technology Co., Ltd. for proteomics determination. According to the proteomics data, the protein sequence of the protease BrgM4 that was significantly upregulated in the fermentation supernatant was screened, and the nucleotide sequence corresponding to the sequence (SEQ ID NO: 1) was sent to GenScript Biotech Co., Ltd. for codon optimization. The obtained protease BrgM4 nucleotide sequence is shown in SEQ ID NO: 2. 2. Heterologous expression and purification of keratinase BrgM4 The codon-optimized nucleotide sequence and pET-28a plasmid (containing 6×His tags at both the C-terminus and the N-terminus) were constructed according to conventional methods to obtain a recombinant plasmid. 10 μL of the recombinant plasmid was transformed into 50 μL of Escherichia coli DH 5α competent cells, and the tube wall was gently tapped to mix. The cells were placed on ice for 30 min, heat-shocked in a 42 ℃ water bath for 90 s, and immediately placed on ice for 3 min. 900 μL of LB liquid culture medium was added to the competent cells, and the cells were incubated at 37 ℃ and 160 rpm for 1 h. After centrifugation at 5000 rpm for 4 min, 500 μL of the supernatant was discarded and gently pipetted to mix. 100 μL was spread on an LB solid plate (containing 50 μg / mL kanamycin) and inverted at 37 ℃ for 12-16 h. The single clone grown on the plate was picked and inoculated into LB liquid culture medium (containing 50 μg / mL kanamycin) and cultured overnight at 37 ℃ and 160 rpm. The clones obtained by culture were used for Star The plasmid was extracted using the Prep Rapid Plasmid Extraction Kit (Beijing Kangrun Chengye Biotechnology Co., Ltd.); The obtained plasmid was transformed into ArcticExpress (DE3) competent cells (Rebit) according to the above method, and the obtained single clone was inoculated into LB liquid medium (containing 50 µg / mL kanamycin) and cultured overnight at 37°C and 160 rpm to prepare seed solution; the seed solution was transferred to fresh LB liquid medium (containing 50 µg / mL kanamycin) at a 1% inoculum amount, and the control group was set as ArcticExpress (DE3) competent cells transformed with pET-28a empty plasmid, and the shaking culture was still carried out at 37°C and 160 rpm. The bacterial mass OD was measured after 2 hours. 600 , when OD 600 When the pH reached the range of 0.6-0.8, IPTG was added to the culture medium (final concentration 0.5 mM) and cultured at 28 °C with shaking at 160 rpm for 12 h; The fermentation broth was centrifuged at 4 °C and 8000 rpm for 10 min, and the fermentation supernatant was discarded to obtain the bacterial cells. After the bacterial cells were suspended with 50 mMTris-HCl buffer (pH 8.0), they were broken by a cell disruptor and centrifuged at 4 °C and 8000 rpm for 10 min to collect the supernatant crude enzyme solution; the crude enzyme solution was purified by a self-assembled nickel column. The purification method is as follows: the nickel column is loaded after equilibration, and gradient elution is performed using 100 mM, 150 mM, 200 mM, and 300 mM imidazole; the eluate is concentrated with a 10 kDa ultrafiltration tube and analyzed by SDS-PAGE to verify the purity and molecular weight of the protein. The results are shown in Figure 1 , the figure shows that the molecular weight of keratinase BrgM4 is about 34 kDa.

[0010] Example 2: Determination of enzymatic properties of keratinase BrgM4 1. Determination of the reaction time of keratinase BrgM4 enzyme activity Before measuring enzyme activity, we must first determine the time range of the initial velocity of the enzymatic reaction. This experiment will use the time and degradation effect curve of the hydrolysis standard substrate (azure keratin) to find the time range of the initial velocity of the enzyme; Using keratin standard substrate azure keratin (Sigma-Aldrich, USA) as substrate, 62.5 µL Tris-HCl buffer (final concentration 50 mM, pH 7.5) and 6.3 µL keratinase prepared in Example 1 (final concentration 0.3 µM) were added to a centrifuge tube containing 2.5 mg azure keratin, and deionized water was added to 250 µL; the centrifuge tube was shaken and incubated at 90 ° C and 1000 rpm for 2, 4, 6, 8, 10, 20, 30, 45, and 60 min, respectively. After centrifugation at 12000 rpm for 10 min, 200 µL of the supernatant was taken to measure its absorbance at 595 nm; a curve was drawn with the enzyme reaction time as the horizontal axis and the absorbance value as the vertical axis, as shown in FIG. Figure 2 The curve shows that when azure keratin is used as the substrate, the slope is the largest within 6 min, that is, the initial velocity time interval of the enzymatic reaction of BrgM4 keratinase is within 6 min, so the reaction time of the enzyme activity determination is set to 5 min; 2. Construction of temperature-pH response surface model Using azure keratin as substrate, 62.5 µL MES buffer (final concentration 50 mM, pH 6.5) and 6.3 µL keratinase (final concentration 0.3 µM) were added to a centrifuge tube containing 2.5 mg azure keratin, and deionized water was added to 250 µL; the centrifuge tube was incubated at 80-100 °C and 1000 rpm for 5 min, and after centrifugation at 12000 rpm for 10 min, 200 µL of the supernatant was taken to measure its absorbance value A at 595 nm. s ; Two reaction systems of pH 7.5 and pH 8.5 Tris-HCl buffer (concentration 50mM) were set up at the same time, and the absorbance value A at 595 nm was also measured. s ; The keratinase prepared in Example 1 was replaced with the heat-inactivated keratinase as a blank control, and the absorbance measured was A b , the final absorbance Δ A=A s -A b .Will ΔSubstitute A into the Beer-Lambert law to calculate the activity of keratinase (A=εlc, A is absorbance, ε is molar absorption coefficient, l is optical path length, c is concentration). According to the data, the molar absorption coefficient of Azure dye used in azure keratin is 135000L / (mol•cm). MODDE 13.1 software was used to design a response surface experiment on the effect of temperature and pH on enzyme activity. A total of 12 different experimental combinations were generated, with pH 7.5 and 90 ℃ as the repeated center points. The response surface model is shown in Figure 3 The model predicted that the optimum temperature was 90 °C, the optimum pH was 7.5, the activity was 0.46 µM / min, and R 2 It is 0.957. It has been verified that under this reaction condition, the measured enzyme activity of keratinase BrgM4 is 0.48 µM / min, indicating that the reliability of the model is high; 3. Thermal stability determination The keratinase prepared in Example 1 was incubated at 60°C, 65°C, 70°C, 75°C, and 80°C for different time periods, and then 62.5 µL of Tris-HCl buffer (final concentration 50 mM, pH 7.5) and 6.3 µL of the incubated keratinase (final concentration 0.3 µM) were added to a centrifuge tube containing 2.5 mg of azure keratin, and deionized water was added to 250 µL; the centrifuge tube was shaken at 90°C and 1000 rpm for 5 min, and after centrifugation at 12000 rpm for 10 min, 200 µL of the supernatant was taken to determine its absorbance at 595 nm, and the keratinase without heat incubation was used as a control, and the enzyme activity was calculated using the Beer-Lambert law; The results showed that after incubation at 60 ℃ for 48 h, the activity of keratinase had no significant difference compared with the control. After incubation at 65 ℃ for 13.62 h, the activity of keratinase was only reduced to half of the original. However, when the incubation temperature was 70 ℃ and 80 ℃, the half-life was 25.77 min and 3.68 min, respectively, indicating that BrgM4 keratinase had a long-term thermal stability at 65 ℃, and its thermal stability gradually decreased with the increase of temperature; 4. Enzyme kinetics assay Using azure keratin as substrate, 62.5 µL Tris-HCl buffer (final concentration 50 mM, pH 7.5) and 6.3 µL keratinase (final concentration 0.3 µM) were added to centrifuge tubes containing different concentrations of azure keratin (8 mg / mL, 12 mg / mL, 18 mg / mL, 24 mg / mL, 30 mg / mL, 40 mg / mL, 60 mg / mL, 70 mg / mL), and deionized water was added to 250 µL; the centrifuge tube was shaken at 90 °C, 1000 rpm for 5 min, and after centrifugation at 12000 rpm for 10 min, 200 µL of the supernatant was taken to measure its absorbance at 595 nm. The enzyme activity was calculated using the Beer-Lambert law. The nonlinear fitting curve was drawn using Origin 2018 and the kinetic constant K was calculated. m and V max value (Michaelis-Menten equation), using the formula k cat =V max / [E]Get k cat Value, fitting curve as Figure 4 As shown, the calculation results show that the K m is 55.20 mg / mL, V max 3.11µM / min, k cat 10.37 min -1 .

[0011] Example 3: Experiment on the degradation of chicken feathers by keratinase BrgM4 1. Preparation of soluble protein standard curve The BCA method was used to determine the concentration of soluble protein (P0012, Beyotime). The concentration of standard soluble protein was set to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. 20 μL of the standard of corresponding concentration was added to each well of a 96-well plate. 200 μL of BCA working solution was added to each well and the plate was placed at 37 °C for 30 min. A was measured using an enzyme reader. 562 , with concentration as the horizontal axis, A 562 As the ordinate, a standard curve was prepared to obtain the linear equation Y=0.3885X+0.2005 (R 2 =0.9937); 2. Experiment of enzymatic hydrolysis of chicken feathers by keratinase BrgM4 and determination of soluble protein content 500 µL Tris buffer (final concentration 50 mM, pH 7.5) and 168 µL BrgM4 (final concentration 1 µM) were added to 20 mg chicken feathers (the chicken feathers were washed with deionized water and sheared to about 5 mm by a grinder), and the deionized water was added to 2 mL; the centrifuge tube was shaken and incubated at 60 °C and 200 rpm for 12 h, 24 h, and 48 h, respectively, and centrifuged at 12000 rpm for 10 min. 20 µL of the supernatant was taken and the A was determined using an enzyme-labeled instrument. 562 , A 562 Substitute into the linear equation in step 1 to obtain the soluble protein content; Results Figure 5 , the proportion of solid chicken feathers converted into soluble protein increased significantly from 12h, and after 48h of enzymatic hydrolysis, the soluble protein content reached 0.41 mg / mL; At the same time, the effect of degrading chicken feathers at 50 °C and 200 rpm was also tested. The method was the same as above, except that MES buffer (concentration 50 mM, pH 6.5) was used and incubated with shaking for 12 h, 24 h, and 48 h. The results are shown in Figure 6 As the enzymatic hydrolysis time increased, the proportion of solid chicken feathers converted into soluble protein increased significantly. After 48 h of enzymatic hydrolysis, the soluble protein content reached 8.87 mg / mL, which was much higher than the soluble protein release at 60 ℃ and pH 7.5. The above results show that keratinase BrgM4 can convert chicken feathers into soluble proteins under high temperature conditions. The present invention provides a treatment approach for the degradation of keratin waste.

Claims

1. A heat-resistant keratinase, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. Use of the heat-resistant keratinase according to claim 1 in degrading chicken feathers.

3. The use according to claim 2, characterized in that: Degrade chicken feathers at 50-80 ℃.

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